Organic compounds

ABSTRACT

The invention relates to novel inhibitors of phosphodiesterase 1 (PDE1), useful for the treatment of diseases or disorders characterized by disruption of or damage to certain cGMP/PKG mediated pathways (e.g., in cardiac tissue). The invention further relates to pharmaceutical composition comprising the same and methods of treatment of cardiovascular disease and related disorders, e.g., congestive heart disease, atherosclerosis, myocardial infarction, and stroke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/034,578, filed on Aug. 7, 2014, the contents of whichare hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to novel inhibitors of phosphodiesterase 1 (PDE1)useful for the treatment of diseases or disorders characterized bydisruption of or damage to certain cGMP/PKG mediated pathways (e.g., incardiac tissue or in vascular smooth muscle). The invention furtherrelates to pharmaceutical composition comprising the same and methods oftreatment of cardiovascular disease and related disorders, e.g.,congestive heart disease, atherosclerosis, myocardial infarction, andstroke.

BACKGROUND OF THE INVENTION

Heart disease is a chronic and progressive illness that kills more than2.4 million Americans each year. There are approximately 500,000 newcases of heart failure per year, with an estimated 5 million patients inthe United States alone having this disease. Early intervention islikely to be most effective in preserving cardiac function. It would bemost desirable to prevent as well to reverse the morphological,cellular, and molecular remodeling that is associated with heartdisease. Some of the most important indicators of cardiac risk are age,hereditary factors, weight, smoking, blood pressure, exercise history,and diabetes. Other indicators of cardiac risk include the subject'slipid profile, which is typically assayed using a blood test, or anyother biomarker associated with heart disease or hypertension. Othermethods for assaying cardiac risk include, but are not limited to, anEKG stress test, thallium stress test, EKG, computed tomography scan,echocardiogram, magnetic resonance imaging study, non-invasive andinvasive arteriogram, and cardiac catheterization.

Pulmonary hypertension (PH or PHT) is an increase in blood pressure inthe pulmonary artery, pulmonary vein, and/or pulmonary capillaries. Itis a very serious condition, potentially leading to shortness of breath,dizziness, fainting, decreased exercise tolerance, heart failure,pulmonary edema, and death. It can be one of five different groups,classified by the World Health Organization in categories describedbelow.

WHO Group I—Pulmonary arterial hypertension (PAH):

-   -   a. Idiopathic (IPAH)    -   b. Familial (FPAH)    -   c. Associated with other diseases (APAH): collagen vascular        disease (e.g. scleroderma), congenital shunts between the        systemic and pulmonary circulation, portal hypertension, HIV        infection, drugs, toxins, or other diseases or disorder.    -   d. Associated with venous or capillary disease.

Pulmonary arterial hypertension involves the vasoconstriction ortightening of blood vessels connected to and within the lungs. Thismakes it harder for the heart to pump blood through the lungs, much asit is harder to make water flow through a narrow pipe as opposed to awide one. Over time, the affected blood vessels become both stiffer andthicker, in a process known as fibrosis. This further increases theblood pressure within the lungs and impairs their blood flow. Inaddition, the increased workload of the heart causes thickening andenlargement of the right ventricle, making the heart less able to pumpblood through the lungs, causing right heart failure. As the bloodflowing through the lungs decreases, the left side of the heart receivesless blood. This blood may also carry less oxygen than normal.Therefore, it becomes more and more difficult for the left side of theheart to pump to supply sufficient oxygen to the rest of the body,especially during physical activity.

WHO Group II—Pulmonary hypertension associated with left heart disease:

-   -   a. Atrial or ventricular disease    -   b. Valvular disease (e.g. mitral stenosis)

In pulmonary hypertension WHO Group II, there may not be any obstructionto blood flow in the lungs. Instead, the left heart fails to pump bloodefficiently out of the heart into the body, leading to pooling of bloodin veins leading from the lungs to the left heart (congestive heartfailure or CHF). This causes pulmonary edema and pleural effusions. Thefluid build-up and damage to the lungs may also lead to hypoxia andconsequent vasoconstriction of the pulmonary arteries, so that thepathology may come to resemble that of Group I or III.

WHO Group III—Pulmonary hypertension associated with lung diseasesand/or hypoxemia:

-   -   a. Chronic obstructive pulmonary disease (COPD), interstitial        lung disease (ILD)    -   b. Sleep-disordered breathing, alveolar hypoventilation    -   c. Chronic exposure to high altitude    -   d. Developmental lung abnormalities

In hypoxic pulmonary hypertension (WHO Group III), the low levels ofoxygen may cause vasoconstriction or tightening of pulmonary arteries.This leads to a similar pathophysiology as pulmonary arterialhypertension.

WHO Group IV—Pulmonary hypertension due to chronic thrombotic and/orembolic disease:

-   -   a. Pulmonary embolism in the proximal or distal pulmonary        arteries    -   b. Embolization of other matter, such as tumor cells or        parasites

In chronic thromboembolic pulmonary hypertension (WHO Group IV), theblood vessels are blocked or narrowed with blood clots. Again, thisleads to a similar pathophysiology as pulmonary arterial hypertension.

WHO Group V—Miscellaneous

Treatment of pulmonary hypertension has proven very difficult.Antihypertensive drugs that work by dilating the peripheral arteries arefrequently ineffective on the pulmonary vasculature. For example,calcium channel blockers are effective in only about 5% of patients withIPAH. Left ventricular function can often be improved by the use ofdiuretics, beta blockers, ACE inhibitors, etc., or by repair/replacementof the mitral valve or aortic valve. Where there is pulmonary arterialhypertension, treatment is more challenging, and may include lifestylechanges, digoxin, diuretics, oral anticoagulants, and oxygen therapy areconventional, but not highly effective. Newer drugs targeting thepulmonary arteries, include endothelin receptor antagonists (e.g.,bosentan, sitaxentan, ambrisentan), phosphodiesterase type 5 inhibitors(e.g., sildenafil, tadalafil), prostacyclin derivatives (e.g.,epoprostenol, treprostenil, iloprost, beroprost), and soluble guanylatecyclase (sGC) activators (e.g., cinaciguat and riociguat). Surgicalapproaches to PAH include atrial septostomy to create a communicationbetween the right and left atria, thereby relieving pressure on theright side of the heart, but at the cost of lower oxygen levels in blood(hypoxia); lung transplantation; and pulmonary thromboendarterectomy(PTE) to remove large clots along with the lining of the pulmonaryartery. Heart failure and acute myocardial infarction are common andserious conditions frequently associated with thrombosis and/or plaquebuild-up in the coronary arteries.

Cardiovascular disease or dysfunction may also be associated withdiseases or disorders typically thought of as affecting skeletal muscle.One such disease is Duchenne muscular dystrophy (DMD), which is adisorder that primarily affects skeletal muscle development but can alsoresult in cardiac dysfunction and cardiomyopathy. DMD is a recessiveX-linked form of muscular dystrophy, affecting around 1 in 3,600 boys,which results in muscle degeneration and eventual death. The disorder iscaused by a mutation in the dystrophin gene, located on the human Xchromosome, which codes for the protein dystrophin, an importantstructural component within muscle tissue that provides structuralstability to the dystroglycan complex (DGC) of the cell membrane. Whileboth sexes can carry the mutation, females rarely exhibit signs of thedisease.

Patients with DMD either lack expression of the protein dystrophin orexpress inappropriately spliced dystrophin, as a result of mutations inthe X-linked dystrophin gene. Additionally, the loss of dystrophin leadsto severe skeletal muscle pathologies as well as cardiomyopathy, whichmanifests as congestive heart failure and arrhythmias. The absence of afunctional dystrophin protein is believed to lead to reduced expressionand mis-localization of dystrophin-associated proteins includingNeuronal Nitric Oxide (NO) Synthase (nNOS). Disruption of nNOS signalingmay result in muscle fatigue and unopposed sympathetic vasoconstrictionduring exercise, thereby increasing contraction-induced damage indystrophin-deficient muscles. The loss of normal nNOS signaling duringexercise is central to the vascular dysfunction proposed to be animportant pathogenic mechanism in DMD. Eventual loss of cardiac functionoften leads to heart failure in DMD patients.

Currently, there is a largely unmet need for an effective way oftreating cardiovascular disease and disorders (e.g. congestive heartdisease), and diseases and disorders which may result in cardiacdysfunction or cardiomyopathy (e.g., Duchenne Muscular Dystrophy).Improved therapeutic compounds, compositions and methods for thetreatment of cardiac conditions and dysfunction are urgently required.

Eleven families of phosphodiesterases (PDEs) have been identified butonly PDEs in Family I, the Ca^(2±)-calmodulin-dependentphosphodiesterases (CaM-PDEs), which are activated by theCa^(2±)-calmodulin and have been shown to mediate the calcium and cyclicnucleotide (e.g. cAMP and cGMP) signaling pathways. The three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in centralnervous system tissue. PDE1A is expressed throughout the brain withhigher levels of expression in the CA1 to CA3 layers of the hippocampusand cerebellum and at a low level in the striatum. PDE1A is alsoexpressed in the lung and heart. PDE1B is predominately expressed in thestriatum, dentate gyrus, olfactory tract and cerebellum, and itsexpression correlates with brain regions having high levels ofdopaminergic innervation. Although PDE1B is primarily expressed in thecentral nervous system, it is also detected in the heart, is present inneutrophils and has been shown to be involved in inflammatory responsesof this cell. PDE1C is expressed in olfactory epithelium, cerebellargranule cells, striatum, heart, and vascular smooth muscle. PDE1C is amajor phosphodiesterase in the human cardiac myocyte.

Of all of the PDE families, the major PDE activity in the human cardiacventricle is PDE1. Generally, there is a high abundance of PDE1 isoformsin: cardiac myocytes, vascular endothelial cells, vascular smooth musclecells, fibroblasts and motor neurons. Up-regulation of phosphodiesterase1A expression is associated with the development of nitrate tolerance.Kim et al., Circulation 104(19:2338-2343 (2001). Cyclic nucleotidephosphodiesterase 1C promotes human arterial smooth muscle cellproliferation. Rybalkin et al., Circ. Res. 90(2):151-157 (2002). Thecardiac ischemia-reperfusion rat model also shows an increase in PDE1activity. Kakkar et al., can. J. Physiol. Pharmacol. 80(1):59-66 (2002).Ca²⁺/CaM-stimulated PDE1, particularly PDE1A has been shown to beinvolved in regulating pathological cardiomyocyte hypertrophy. Millet etal., Circ. Res. 105(10):956-964 (2009). Early cardiac hypertrophyinduced by angiotensin II is accompanied by 140% increases in PDE1A in arat model of heart failure. Mokni et al., Plos. One. 5(12):e14227(2010). Inhibition of phosphodiesterase 1 augments the pulmonaryvasodilator response to inhaled nitric oxide in awake lambs with acutepulmonary hypertension. Evgenov et al., Am. J. Physiol. Lung Cell. Mol.Physiol. 290(4):L723-L729 (2006). Strong upregulation of the PDE1 familyin pulmonary artery smooth muscle cells is also noted in humanidiopathic PAH lungs and lungs from animal models of PAH. Schermuly etal., Circulation 115(17)2331-2339 (2007). PDE1A and 1C, found infibroblasts, are known to be up-regulated in the transition to the“synthetic phenotype”, which is connected to the invasion of diseasedheart tissue by pro-inflammatory cells that will deposit extracellularmatrix. PDE1B2, which is present in neutrophils, is up-regulated duringthe process of differentiation of macrophages. Bender et al., PNAS102(2):497-502 (2005). The differentiation of monocytes to macrophage,in turn, is involved in the inflammatory component of heart disease,particularly atherothrombosis, the underlying cause of approximately 80%of all sudden cardiac death. Willerson et al., Circulation109:II-2-II-10 (2004).

Cyclic nucleotide phosphodiesterases downregulate intracellular cAMP andcGMP signaling by hydrolyzing these cyclic nucleotides to theirrespective 5′-monophosphates (5′AMP and 5′GMP). cGMP is a centralintracellular second-messenger regulating numerous cellular functions.In the cardiac myocyte, cGMP mediates the effects of nitric oxide andatrial natriuretic peptide, whereas its counterpart, cAMP, mediatescatecholamine signaling. Each cyclic nucleotide has a correspondingprimary targeted protein kinase, PKA for cAMP, and PKG for cGMP. PKAstimulation is associated with enhanced contractility and can stimulategrowth, whereas PKG acts as a brake in the heart, capable of counteringcAMP-PKA-contractile stimulation and inhibiting hypertrophy.Importantly, the duration and magnitude of these signaling cascades aredetermined not only by generation of cyclic nucleotides, but also bytheir hydrolysis catalyzed by phosphodiesterases (PDEs). PDE regulationis quite potent—often suppressing an acute rise in a given cyclicnucleotide back to baseline within seconds. It is also compartmentalizedwithin the cell, so that specific targeted proteins can be regulated bythe same “generic” cyclic nucleotide. By virtue of its modulation ofcGMP in the myocyte, PDE1 participates in hypertrophy regulation. (CircRes. 2009, Nov. 6; 105(10):931).

One of the challenges currently faced in the field is the lack of PDE1specific inhibitors. The current invention seeks to overcome this aswell as other challenges in the art by providing PDE1 specificinhibitors. Although WO 2006/133261 and WO 2009/075784 provide PDE1specific inhibitors, these do not disclose the compounds of the currentinvention.

SUMMARY OF THE INVENTION

PDE1 is up-regulated in chronic disease conditions such asatherosclerosis, cardiac pressure-load stress and heart failure, as wellas in response to long-term exposure to nitrates. PDE1 inhibitors haverelatively little impact on resting function, but rather maintain theability to potently modulate acute contractile tone in cells stimulatedby vasoactive agonists. Such up-regulation contributes to vascular andcardiac pathophysiology and to drug tolerance to nitrate therapies.Therefore, without being bound by theory, it is believed that compoundsthat modulate cGMP/PKG mediated pathways, such as PDE1 inhibitors, areparticularly useful for reversing cardiac hypertrophy. The PDE1inhibitors disclosed herein are selective PDE1 inhibitors having alimited ability to penetrate the blood brain barrier and therefore, arebelieved to have significant modulatory activity (e.g., enhancement ofcGMP) in those areas of the body outside of the central nervous systemwhere PDE1 isoforms are predominately located: e.g., cardiac, vascular,and lung tissue.

Therefore, in the first aspect, the invention provides a compound ofFormula I:

in free or salt form, wherein

-   (i) R₁ is —NH(R₄), wherein R₄ is phenyl optionally substituted with    halo (e.g., fluoro), for example, 4-fluorophenyl;-   (ii) R₂ is H or C₁₋₆ alkyl (e.g., methyl, isobutyl or neopentyl);-   (iii) R₃ is —SO₂NH₂ or —COOH;    in free or salt form.

In a particular embodiment, the invention provides a compound of FormulaI as follows:

-   -   1.1 the compound of Formula I, wherein R₁ is —NH(R₄) wherein R₄        is phenyl optionally substituted with halo (e.g., fluoro), for        example, 4-fluorophenyl;    -   1.2 the compound of Formula I or 1.1, wherein R₄ is phenyl;    -   1.3 the compound of Formula I or 1.1, wherein R₄ is        4-fluorophenyl;    -   1.4 the compound of Formula I or any of Formulas 1.1-1.3,        wherein R₂ is C₁₋₆alkyl (e.g., methyl, isobutyl or neopentyl);    -   1.5 the compound of Formula I or any of Formulas 1.1-1.4,        wherein R₂ is methyl;    -   1.6 the compound of Formula I or any of Formulas 1.1-1.4,        wherein R₂ is isobutyl;    -   1.7 the compound of Formula I or any of Formulas 1.1-1.4,        wherein R₂ is neopentyl;    -   1.8 the compound of Formula I or any of Formulas 1.1-1.7,        wherein R₃ is —SO₂NH₂ or —COOH;    -   1.9 the compound of Formula I or any of Formulas 1.1-1.8,        wherein R₃ is —SO₂NH₂;    -   1.10 the compound of Formula I or any of Formulas 1.1-1.8,        wherein R₃ is —COOH;    -   1.11 the compound of Formula I or any of Formulas 1.1-1.10,        wherein the compound is:

-   -   1.12 the compound of Formula I or any of Formulas 1.1-1.10,        wherein the compound is:

-   -   1.13 the compound of Formula I or any of Formulas 1.1-1.10,        wherein the compound is:

in free or salt form.

In the second aspect, the invention provides a pharmaceuticalcomposition comprising the compound of Formula I or any of 1.1-1.13 asdescribed herein, in free or pharmaceutically acceptable salt form, inadmixture with a pharmaceutically acceptable diluents or carrier.

The compound of Formula I or any of 1.1-1.13 as described herein areselective PDE1 inhibitors and therefore are useful for regulatingcGMP/PKG in cardiac hypertrophy. Previous studies have demonstrated thatincreases in intracellular Ca²⁺/CaM-dependent signaling promotemaladaptive hypertrophic gene expression in cardiomyocytes throughvarious effectors such as the protein phosphatase calcineurin,Ca²⁺/CaM-dependent kinase II (CaMKII). Without being bound by anytheory, increases in endogenous cGMP/PKG-dependent signaling may be ableto decrease cardiac hypertrophy, by suppressing Gq/11 activation andnormalizing Ca²⁺ signaling. By activating PDE1, Ca²⁺/CaM may decreasecGMP levels and PKG activity. In turn, this process may drive cardiachypertrophy. Additionally, up-regulation of PDE1 expression uponneurohumoral or biomechanical stress during cardiac hypertrophy mayfurther enhance PDE1 activity and attenuate cGMP/PKG signaling.Accordingly, without being bound by any theory, it is believed thatinhibition of PDE1A could, for example, reverse or prevent theattenuation of cGMP/PKG signaling. As discussed previously, PDE1B isalso implicated in the inflammatory component of heart disease (e.g.,PDE1B2 is up-regulated during the process of differentiation ofmacrophages, as occurs during heart disease progression). Similarly,PDE1C is induced in human arterial smooth muscle cells of the synthetic,proliferative phenotype. Therefore, administration of a PDE1 inhibitoras described herein could provide a potential means to regulate cardiachypertrophy, and by extension provide a treatment for variouscardiovascular diseases and disorders.

Therefore, in the third aspect, the invention provides a method for thetreatment or prophylaxis of a disease or disorder which may beameliorated by modulating (e.g., enhancing) cGMP/PKG-dependent signalingpathways (e.g., in cardiac tissue), e.g. a cardiovascular disease ordisorder, comprising administering to a patient in need thereof aneffective amount of the compound of Formula I or any of formulae1.1-1.13 as described herein, in free or pharmaceutically acceptablesalt form.

The cardiovascular disease or disorder may be selected from a groupconsisting of: hypertrophy (e.g., cardiac hypertrophy), atherosclerosis,myocardial infarction, congestive heart failure, angina, stroke,hypertension, essential hypertension, pulmonary hypertension, pulmonaryarterial hypertension, secondary pulmonary hypertension, isolatedsystolic hypertension, hypertension associated with diabetes,hypertension associated with atherosclerosis, renovascular hypertension.In certain embodiments, the cardiovascular disease or disorder to betreated may also relate to impaired cGMP/PKG-dependent signaling. In aparticular embodiment, the invention provides a method for the treatmentor prevention of stroke, wherein the PDE1 inhibitor, through its effecton the endothelial cells of cerebral capillaries, is able to promoteincreased cerebral blood flow.

In a further embodiment of the third aspect, the invention also providesa method for the treatment or prophylaxis of cardiovascular disease ordisorder that is associated with a muscular dystrophy (e.g, Duchenne,Becker, limb-girdle, myotonic, and Emery-Dreifuss Muscular Dystrophy)comprising administering to a patient in need thereof an effectiveamount of the compound of Formula I or any of 1.1-1.13 as describedherein, in free or pharmaceutically acceptable salt form. As notedabove, DMD is caused by the absence of a functional dystrophin protein,which in turn leads to reduced expression and mis-localization ofdystrophin-associated proteins, such as neuronal nitric oxide (NO)synthase. Disruption of nNOS signaling may result in muscle fatigue andunopposed sympathetic vasoconstriction during exercise, therebyincreasing contraction-induced damage in dystrophin-deficient muscles.Without being bound by theory, the loss of normal nNOS signaling duringexercise may be central to the vascular dysfunction proposed to be animportant pathogenic mechanism in DMD. It is contemplated that byinhibiting phosphodiesterases (e.g. PDE1), the compounds describedherein may circumvent defective nNOS signaling in dystrophic skeletaland/or cardiac muscle; thereby potentially improving cardiac outcomes inDMD patients.

In still another embodiment, the invention provides for the treatment ofrenal failure, fibrosis, inflammatory disease or disorders, vascularremodeling and connective tissue diseases or disorders (e.g., MarfanSyndrome), comprising administering to a patient in need thereof aneffective amount of the compound of Formula I or any of formulae1.1-1.13 as described herein, in free or pharmaceutically acceptablesalt form.

The PDE1 compounds of the invention useful for the treatment orprophylaxis of disease according to the foregoing methods may be used asa sole therapeutic agent or may be used in combination with one or moreother therapeutic agents useful for the treatment of cardiovasculardisorders. Such other agents include angiotensin II receptorantagonists, angiotensin-converting-enzyme (ACE) inhibitors, neutralendopeptidase (NEP or Neprilysin) inhibitors and/or phosphodiesterase 5(PDE5) inhibitors.

Therefore, in a particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with an angiotensin IIreceptor antagonist selected from azilsartan, candesartan, eprosartan,irbesartan, losartan, olmesartan, olmesartan medoxomil, saralasin,telmisartan and valsartan, in free or pharmaceutically acceptable saltform.

In yet another embodiment, the PDE1 inhibitor of the invention may beadministered in combination with an angiotensin-converting-enzyme (ACE)inhibitor selected from the group consisting of: captopril, enalapril,lisinopril, benazapril, ramipril, quinapril, peridopril, imidapril,trandolapril and cilazapril, in free or pharmaceutically acceptable saltform.

In still another particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with a PDE5 inhibitorselected from avanafil, lodenafil, mirodenafil, tadalafil, vardenafil,udenafil and zaprinast, in free or pharmaceutically acceptable saltform.

In still another particular embodiment, the PDE1 inhibitor of theinvention may be administered in combination with a neutralendopeptidase (NEP or Neprilysin) inhibitor. Neutral endopeptidase, alsoknown as Neprilysin or NEP (EC 3.4.24.11), is a type II integralmembrane zinc-dependent metalloendoprotease that cleaves a variety ofshort peptide substrates. Among its natural targets are cardiac atrialnatriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-typenatriuretic peptide (CNP), angiotensin I (Ang-I), angiotensin II(Ang-II), bradykinin (BK), and endothelin (ET). Cleavage of thesepeptides by NEP results in their inactivation, attenuating the peptides'natural biological effects.

ANP, BNP and CNP are all part of the natriuretic peptide (NP) system,which, along with the renin-angiotensin system, is a major component ofmammalian blood pressure homeostasis. While the renin-angiotensin systemis primarily responsible for increasing blood pressure (e.g., bypromoting vasoconstriction and water retention), the natriuretic peptidesystem is primarily responsible for decreasing blood pressure (e.g., bypromoting vasodilation and natriuresis). ANP and BNP are both powerfulvasodilators and strong promoters of decreased renal reabsorption ofsodium and water in a potassium-sparing manner. These dual effects exerta powerful blood pressure lowering effect. BNP and CNP also exert ananti-fibrotic effect and an anti-hypertrophic effect in the heart. CNPshares the vasodilatory effects of ANP/BNP but without the renaleffects. In addition, both hypertension and obesity have been shown tobe associated with reduced ANP and BNP levels, and a specific geneticvariant of ANP (rs5068), which increases ANP levels, has been shown toprotect against hypertension and metabolic syndrome. Thus, ANP, BNP andCNP play an important role in blood pressure homeostasis andcardiovascular health.

Inhibition of NEP results in an increase in the half-lives ofcirculating ANP, BNP and CNP. This is expected to prolong theirblood-pressure lowering and cardiac health improving effects. Urine cAMPlevels ae significantly elevated after systemic administration of NEPinhibitors.

Inhibition of NEP also results in higher levels of bradykinin,angiotensin I, angiotensin II and endothelin. Importantly, endothelinand angiotensin II are strongly pro-hypertensive peptides. Thus, NEPinhibition alone results in both vasodilatory effects (from the NPs) andvasoconstrictive effects (from increased Ang-II and ET). Thesepro-hypertensive peptides all operate via binding to G-protein coupledreceptors (GPCRs). The major contributor to this vasoconstrictive effectis Angiotensin-II, which operates via binding to the G-protein coupledreceptors AT₁ and AT₂. These receptors exert their physiological effectsthrough activation of phospholipase C (PLC) and protein kinase C (PKC)signaling cascades. Bradykinin is inactivated to a large extent by ACE,and ACE inhibitors cause congestion as a major side effect, which is notseen with NEP inhibitors.

ANP, BNP and CNP all function via the second messenger cGMP. ANP and BNPbind to membrane-bound guanylyl cyclase-A, while CNP binds to guanylylcyclase B. Both of these enzymes increase intracellular cGMP in responseto receptor binding. The increased cGMP concentration activates proteinkinase G (PKG) which is responsible for exerting the downstreambiological effects of the natriuretic peptides.

Several NEP inhibitors are known, including candoxatril, candoxatrilat,omepatrilat, gempatrilat, and sampatrilat. Candoxtrail had been shown toproduce a dose-dependent increase in both plasma ANP and cGMP levels,and although it is safe, it does not produce a stable blood-pressurelowering effect. This is thought to be due to the effects of NEPinhibition on BK, ET and Ang-II breakdown. Candoxatril treatment inpatients with heart failure has been shown to increase levels ofendothelin significantly, thus cancelling out the blood pressure effectscaused by increased ANP.

In contrast to candoxatril and candoxatrilat, omapatrilat is considereda vasopeptidase inhibitor (VPI), because it functions to an equal extentas both an NEP inhibitor and an ACE (angiotensin converting enzyme)inhibitor. ACE is the enzyme that is responsible for converting Ang-I toAng-II, which is the major pro-hypertensive hormone of therenin-angiotensin system. By both inhibiting NEP and ACE, it was thoughtthat the increase in Ang-II caused by NEP inhibition would be negated,resulting in a highly effective antihypertensive treatment. Clinicalstudies, however, showed that omapatrilat was associated with a severeincidence of angioedema (a known side effect of ACE inhibitors). Laterresearch has indicated that this may be due to concomitant inhibition ofaminopeptidase P (APP). ACE, APP and NEP all contribute to the breakdownof bradykinin, which is another anti-hypertensive peptide, and theover-accumulation of bradykinin resulting from simultaneous inhibitionof three of its degradation pathways may be a strong factor leading toangioedema.

Without intending to be bound by any particular theory, the combinationof a PDE1 inhibitor with a selective NEP inhibitor (not a VPI) shouldrealize the full positive effects of NEP inhibition (increased ANP, BNPand CNP half-life), further enhanced by the potentiation of the NPsignaling cascades (mediated by cGMP) caused by PDE1 inhibition, withoutthe negative effects of NEP inhibition that can lead to decreasedefficacy.

Therefore, in a particular embodiment, the neutral endopeptidase (NEP orNeprilysin) inhibitor useful for the invention is a selective NEPinhibitor, e.g., with at least 300-fold selectivity for NEP inhibitionover ACE inhibition. In a further embodiment, the NEP inhibitors for usein the current invention are inhibitors with at least 100-foldselectivity for NEP inhibition over ECE (Endothelin Converting Enzyme)inhibition. In yet another embodiment, the NEP inhibitors for use in thecurrent invention are inhibitors with at least 300-fold selectivity forNEP inhibition over ACE inhibition and 100-fold selectivity for NEPinhibition over ECE inhibition.

In another embodiment, the NEP inhibitors for use in the currentinvention are the NEP inhibitors disclosed in the followingpublications: EP-1097719 B1, EP-509442A, U.S. Pat. No. 4,929,641,EP-599444B, US-798684, J. Med. Chem. (1993) 3821, EP-136883, US-4722810,Curr. Pharm. Design (1996) 443, J. Med. Chem. (1993) 87, EP-830863,EP-733642, WO 9614293, WO 9415908, WO 9309101, WO 9109840, EP-519738,EP-690070, Bioorg. Med. Chem. Lett. (1996) 65, EP-A-0274234, Biochem.Biophys. Res. Comm. (1989) 58, Perspect. Med. Chem. (1993) 45, orEP-358398-B. The contents of these patents and publications are herebyincorporated by reference in their entirety herein.

In still another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors Phosphoramidon, Thiorphan,Candoxatrilat, Candoxatril, or the compound of the Chemical AbstractService (CAS) Number 115406-23-0.

In yet another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in US 2006/0041014 A1, thecontents of which are hereby incorporated by reference in their entiretyherein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 5,217,996,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 8,513,244,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in U.S. Pat. No. 5,217,996,the contents of which are hereby incorporated by reference in theirentirety herein.

In another embodiment, the NEP inhibitors useful in the currentinvention are the NEP inhibitors disclosed in US patent applicationpublication 2013/0330365, the contents of which are hereby incorporatedby reference in their entirety herein.

In another embodiment, the NEP inhibitor useful in the current inventionis3-[{1S,5R}-1-biphenyl-4ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl]propionicacid,

also known as AHU-377, in free or a pharmaceutically acceptable salt orprodrug, thereof, and in a preferred embodiment thereof, in sodium saltform.

In another embodiment, the NEP inhibitor useful in the current inventionis [2R,4R}-1-biphenyl-4ylmethyl-3-carboxy-1-butylcarbamoyl]propionicacid,

also known as LBQ-657, in free or pharmaceutically acceptable ester,salt or prodrug form.

In another embodiment, the NEP inhibitor useful in the current inventionis selected from among the following: sampatrilat, fasidotril, Z13752A,MDL 100240, BMS 189921, LBQ657, AHU-377, or mixanpril, in free orpharmaceutically acceptable salt form or in prodrug form thereof.

In another embodiment, the NEP inhibitor for use in the currentinvention is selected from the following:

-   SQ 28,603;-   N—[N-[1(S)-carboxyl-3-phenylpropyl]-(S)-phenylalanyl]-(S)-isoserine;-   N—[N-[((1S)-carboxy-2-phenyl)ethyl]-(S)-phenylalanyl]beta-alanine;-   N-[2(S)-mercaptomethyl-3-(2-methylphenyl)-propionyl]methionine;-   (cis-4-[[[1-[2-carboxy-3-(2-methoxy-ethoxy)propyl]-cyclopentyl]carbonyI]amino]-cyclohexanecarboxylic    acid);-   thiorphan; retro-thiorphan; phosphoramidon; SQ 29072;-   N-(3-carboxy-1-oxopropyl)-(4S)-p-phenylphenylmethyl)-4-amino-2R-methylbutanoic    acid ethyl ester;-   (S)-cis-4-[1-[2-(5-indanyloxycarbonyl)-3-(2-methoxyethoxy)propyl]-1-cyclopentanecarboxamido]-1-cyclohexanecarboxylic    acid;-   3-(1-[6-endo-hydroxymethyl-bicyclo[2,2,1]heptane-2-exo-carbamoyl]cyclopentyl)-2-(2-methoxyethyl)propanoic    acid;-   N-(1-(3-(N-t-butoxycarbonyl-(S)-prolylamino)-2(S)-t-butoxy-carbonylpropyl)cyclopentanecarbonyl)-O-benzyl-(S)-serine    methyl ester;-   4-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]benzoic acid;-   3-[1-(cis-4-carboxycarbonyl-cis-3-butylcyclohexyl-r-1-carbamoyl)cyclopentyl]-2S-(2-methoxyethoxymethyl)propanoic    acid;-   N-((2S)-2-(4-biphenylmethyl)-4-carboxy-5-phenoxyvaleryl)glycine;-   N-(1-(N-hydroxycarbamoylmethyl)-1-cyclopentanecarbonyl)-L-phenylalanine;-   (S)-(2-biphenyl-4-yl)-1-(1H-tetrazol-5-yl)ethylamino)methylphosphonic    acid;-   (S)-5-(N-(2-(phosphonomethylamino)-3-(4-biphenyl)propionyl)-2-aminoethyl)tetrazole;-   beta-alanine;-   3-[1,1′-biphenyl]-4-yl-N-[diphenoxyphosphinyl)methyl]-L-alanyl;-   N-(2-carboxy-4-thienyl)-3-mercapto-2-benzylpropanamide;-   2-(2-mercaptomethyl-3-phenylpropionamido)thiazol-4-ylcarboxylic    acid;-   (L)-(1-((2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy)carbonyl)-2-phenylethyl)-L-phenylalanyl)-beta-alanine;-   N—[N-[(L)-[1-[(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-(R)-alanine;-   N—[N-[(L)-1-carboxy-2-phenylethyl]-L-phenylalanyl]-(R)-alanine;-   N-[2-acetylthiomethyl-3-(2-methyl-phenyl)propionyl]-methionine ethyl    ester;-   N-[2-mercaptomethyl-3-(2-methylphenyl)propionyl]-methionine;-   N-[2(S)-mercaptomethyl-3-(2-methylphenyl)propanoyl]-(S)-isoserine;-   N—(S)-[3-mercapto-2-(2-methylphenyl)propionyl]-(S)-2-methoxy-(R)-alanine;-   N-[1-[[1(S)-benzyloxy-carbonyl-3-phenylpropyl]amino]cyclopentylcarbony    1]-(S)-isoserine;-   N-[1-[[1(S)-carbonyl-3-phenylpropyl]amino]-cyclopentylcarbonyl]-(S)-isoserine;-   1,1′-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis-(S)-isoserine;-   1,1′-[dithiobis-[2(S)-(2-methylbenzyl)-1-oxo-3,1-propanediyl]]-bis-(S)-methionine;-   N-(3-phenyl-2-(mercaptomethyl)-propionyl)-(S)-4-(methylmercapto)methionine;-   N-[2-acetylthiomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;-   N-[2-mercaptomethyl-3-phenyl-propionyl]-3-aminobenzoic acid;-   N-[1-(2-carboxy-4-phenylbutyI)-cyclopentane-carbonyl]-(S)-isoserine;-   N-[1-(acetylthiomethyl)cyclopentane-carbonyl]-(S)-methionine ethyl    ester;-   3(S)-[2-(acetylthiomethyl)-3-phenyl-propionyl]amino-epsilon-caprolactam;-   N-(2-acetylthiomethy 1-3-(2-methylphenyl)propionyl)-methionine ethyl    ester;    in free or pharmaceutically acceptable salt form.

In another aspect, the invention provides the following:

-   -   (i) the compound of Formula I or any of 1.1-1.13 as described        herein, in free or pharmaceutically acceptable salt form, for        use in any of the methods or in the treatment or prophylaxis of        any disease or disorder as set forth herein,    -   (ii) a combination as described hereinbefore, comprising a PDE1        inhibitor of the invention, e.g., the compound of Formula I or        any of 1.1-1.13 as described herein, in free or pharmaceutically        acceptable salt form and a second therapeutic agent useful for        the treatment of cardiovascular disorders, e.g., selected from        angiotensin II receptor antagonist,        angiotensin-converting-enzyme (ACE) inhibitor, neutral        endopeptidase (NEP or Neprilysin) inhibitor and/or        phosphodiesterase 5 (PDE5) inhibitor, in free or        pharmaceutically acceptable salt form;    -   (iii) use of the compound of Formula I or any of 1.1-1.13, in        free or pharmaceutically acceptable salt form, or the        combination described herein, (in the manufacture of a        medicament) for the treatment or prophylaxis of any disease or        condition as set forth herein,    -   (iv) the compound of Formula I or any of 1.1-1.13, in free or        pharmaceutically acceptable salt form, the combination described        herein or the pharmaceutical composition of the invention as        hereinbefore described for use in the treatment or prophylaxis        of any disease or condition as set forth herein.

DETAILED DESCRIPTION OF THE INVENTION

If not otherwise specified or clear from context, the following termsherein have the following meanings:

-   -   (a) “Alkyl” as used herein is a saturated or unsaturated        hydrocarbon moiety, preferably saturated, preferably having one        to six carbon atoms, in some embodiment, one to four carbon        atoms, which may be linear or branched, and may be optionally        mono-, di- or tri-substituted, e.g., with halogen (e.g., chloro        or fluoro) or hydroxy.

Compounds of the Invention, e.g., the compound of Formula I or any offormulae 1.1-1.13 as described herein, may exist in free or salt form,e.g., as acid addition salts. In this specification unless otherwiseindicated, language such as “Compounds of the Invention” is to beunderstood as embracing the compounds in any form, for example free oracid addition salt form, or where the compounds contain acidicsubstituents, in base addition salt form. The Compounds of the Inventionare intended for use as pharmaceuticals, therefore pharmaceuticallyacceptable salts are preferred. Salts which are unsuitable forpharmaceutical uses may be useful, for example, for the isolation orpurification of free Compounds of the Invention or theirpharmaceutically acceptable salts, are therefore also included.

Compounds of the Invention may in some cases exist in prodrug form. Aprodrug form is compound which converts in the body to a Compound of theInvention. For example when the Compounds of the Invention containhydroxy or carboxy substituents, these substituents may formphysiologically hydrolysable and acceptable esters. As used herein,“physiologically hydrolysable and acceptable ester” means esters ofCompounds of the Invention which are hydrolysable under physiologicalconditions to yield acids (in the case of Compounds of the Inventionwhich have hydroxy substituents) or alcohols (in the case of Compoundsof the Invention which have carboxy substituents) which are themselvesphysiologically tolerable at doses to be administered. Therefore,wherein the Compound of the Invention contains a hydroxy group, forexample, Compound-OH, the acyl ester prodrug of such compound, i.e.,Compound-O—C(O)—C₁₋₄ alkyl, can hydrolyze in the body to formphysiologically hydrolysable alcohol (Compound-OH) on the one hand andacid on the other (e.g., HOC(O)—C₁₋₄ alkyl). Alternatively, wherein theCompound of the Invention contains a carboxylic acid, for example,Compound-C(O)OH, the acid ester prodrug of such compound,Compound-C(O)O—C₁₋₄ alkyl can hydrolyze to form Compound-C(O)OH andHO—C₁₋₄alkyl. As will be appreciated the term thus embraces conventionalpharmaceutical prodrug forms.

The Compounds of the Invention include their enantiomers, diastereomersand racemates, as well as their polymorphs, hydrates, solvates andcomplexes. Some individual compounds within the scope of this inventionmay contain double bonds. Representations of double bonds in thisinvention are meant to include both the E and the Z isomer of the doublebond. In addition, some compounds within the scope of this invention maycontain one or more asymmetric centers. This invention includes the useof any of the optically pure stereoisomers as well as any combination ofstereoisomers.

It is also intended that the Compounds of the Invention encompass theirstable and unstable isotopes. Stable isotopes are nonradioactiveisotopes which contain one additional neutron compared to the abundantnuclides of the same species (i.e., element). It is expected that theactivity of compounds comprising such isotopes would be retained, andsuch compound would also have utility for measuring pharmacokinetics ofthe non-isotopic analogs. For example, the hydrogen atom at a certainposition on the Compounds of the Invention may be replaced withdeuterium (a stable isotope which is non-radioactive). Examples of knownstable isotopes include, but not limited to, deuterium, ¹³C, ¹⁵N, ¹⁸O.Alternatively, unstable isotopes, which are radioactive isotopes whichcontain additional neutrons compared to the abundant nuclides of thesame species (i.e., element), e.g., 123I, ¹³¹I, ¹²⁵I, ¹⁸F, may replacethe corresponding abundant species of I, C and F. Another example ofuseful isotope of the compound of the invention is the ¹¹C isotope.These radio isotopes are useful for radio-imaging and/or pharmacokineticstudies of the compounds of the invention.

Melting points are uncorrected and (dec) indicates decomposition.Temperature are given in degrees Celsius (° C.); unless otherwisestated, operations are carried out at room or ambient temperature, thatis, at a temperature in the range of 18-25° C. Chromatography meansflash chromatography on silica gel; thin layer chromatography (TLC) iscarried out on silica gel plates. NMR data is in the delta values ofmajor diagnostic protons, given in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard. Conventionalabbreviations for signal shape are used. Coupling constants (J) aregiven in Hz. For mass spectra (MS), the lowest mass major ion isreported for molecules where isotope splitting results in multiple massspectral peaks Solvent mixture compositions are given as volumepercentages or volume ratios. In cases where the NMR spectra arecomplex, only diagnostic signals are reported.

Methods of Using Compounds of the Invention

The Compounds of the Invention are useful in the treatment of diseasescharacterized by disruption of or damage to cGMP/PKG mediated pathways,e.g., as a result of increased expression of PDE1 or decreasedexpression of cGMP/PKG activity due to inhibition or reduced levels ofinducers of cyclic nucleotide synthesis, such as dopamine and nitricoxide (NO). It is believed that by inhibiting PDE1A or PDE1C, forexample, that this action could reverse or prevent the attenuation ofcGMP/PKG signaling (e.g., enhance cGMP) and that this action couldmodulate cardiac hypertrophy. Therefore, administration or use of thePDE1 inhibitor as described herein, e.g., a Compound of Formula 1.1-1.13as described herein, in free or pharmaceutically acceptable salt form,could provide a potential means to regulate cardiac hypertrophy (e.g.,prevent and/or reverse cardiac hypertrophy), and in certain embodimentsprovide a treatment for various cardiovascular diseases and disorders.

The PDE1 inhibitors of the present invention generally are selective forPDE1 (generally off-target interactions >10×, more preferably >25×,still more preferably >100× affinity for PDE1), exhibit good oralavailability in plasma with very minimal brain penetration in mice. Theblood/plasma ratio in mice administered the PDE1 inhibitors of thepresent invention is preferably less than 0.4, more preferably less than0.2, more preferably less than or equal to 0.1.

The PDE1 inhibitor of the invention (i.e., Formula I as hereinbeforedescribed) may be used in a combination therapy wherein the PDE1inhibitor may be administered simultaneously, separately or sequentiallywith another active agent. Therefore, the combination can be a freecombination or a fixed combination.

The term “simultaneously” when referring to a therapeutic use meansadministration of two or more active ingredients at or about the sametime by the same route of administration.

The term “separately” when referring to a therapeutic use meansadministration of two or more active ingredients at or about the sametime by different route of administration.

The dosages of a compound of the invention in combination with anotheractive agent can be the same as or lower than the approved dosage forthe drug, the clinical or literature test dosage or the dosage used forthe drug as a monotherapy. The dosage amount will be apparent to oneskilled in the art.

Diseases and disorders that may be prevented or ameliorated by theenhancement of cGMP/PKG signaling (e.g., cardiovascular disease), e.g.,using the Compound of the Invention as described herein, include, butare not limited to: hypertrophy (e.g., cardiac hypertrophy),atherosclerosis, myocardial infarction, congestive heart failure,angina, stroke, hypertension, essential hypertension, pulmonaryhypertension, pulmonary arterial hypertension, secondary pulmonaryhypertension, isolated systolic hypertension, hypertension associatedwith diabetes, hypertension associated with atherosclerosis,renovascular hypertension, renal failure, fibrosis, an inflammatorydisease or disorder, vascular remodeling, and an connective tissuedisease or disorder (e.g., Marfan Syndrome).

In one embodiment, the Compounds of the Invention as described hereinare useful in the treatment or prevention of stroke by treating orpreventing transient ischemic attacks (TIA). Without being bound by anytheory, it is believed that the Compounds of the Invention may preventor treat the risk of transient ischemic attacks by actually increasingthe amount and/or concentration of blood flow to the brain. It iscontemplated that the compounds as described herein could increase theblood flow to the brain without significant passage across the bloodbrain barrier.

In another embodiment, the invention further provides using theCompounds of the Invention for the treatment or prevention of disease ordisorder as follows: Duchenne muscular dystrophy, Becker musculardystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, andEmery-Dreifuss muscular dystrophy. In one embodiment, the Compounds ofthe Invention are useful in treating cardiac dysfunction associated withaforementioned types of muscular dystrophy. In another embodiment, theCompounds of the Invention may potentially reduce or reverse the cardiachypertrophy that may be associated with these aforementioned types ofmuscular dystrophy.

“PDE1 inhibitor” as used herein describes a compound of Formula I or anyof formulae 1.1-1.13. which selectively inhibitphosphodiesterase-mediated (e.g., PDE1-mediated, especiallyPDE1B-mediated) hydrolysis of cGMP, e.g., with an IC₅₀ of less than 1μM, preferably less than 75 nM, preferably less than 1 nM, in animmobilized-metal affinity particle reagent PDE assay as described orsimilarly described in Example 1.

The phrase “Compounds of the Invention” or “PDE 1 inhibitors of theInvention”, or like terms, encompasses any and all of the compoundsdisclosed herewith, e.g., a Compound of Formula I or any of formulae1.1-1.13, in free or salt form.

The words “treatment” and “treating” are to be understood accordingly asembracing treatment or amelioration of symptoms of disease as well astreatment of the cause of the disease.

For methods of treatment, the word “effective amount” is intended toencompass a therapeutically effective amount to treat a specific diseaseor disorder.

The term “patient” include human or non-human (i.e., animal) patient. Inparticular embodiment, the invention encompasses both human andnonhuman. In another embodiment, the invention encompasses nonhuman. Inother embodiment, the term encompasses human.

The term “comprising” as used in this disclosure is intended to beopen-ended and does not exclude additional, unrecited elements or methodsteps.

Compounds of the Invention, e.g., compounds of Formula I or any offormulas 1.1-1.13 as hereinbefore described, in free or pharmaceuticallyacceptable salt form, may be used as a sole therapeutic agent, but mayalso be used in combination or for co-administration with other activeagents.

Dosages employed in practicing the present invention will of course varydepending, e.g. on the particular disease or condition to be treated,the particular compound used, the mode of administration, and thetherapy desired. The compound may be administered by any suitable route,including orally, parenterally, transdermally, or by inhalation, but arepreferably administered orally. In general, satisfactory results, e.g.for the treatment of diseases as hereinbefore set forth are indicated tobe obtained on oral administration at dosages of the order from about0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicateddaily dosage for oral administration will accordingly be in the range offrom about 0.75 to 300 mg, conveniently administered once, or in divideddoses 2 to 4 times, daily or in sustained release form. Unit dosageforms for oral administration thus for example may comprise from about0.2 to 75 or 150 mg or 300 mg, e.g. from about 0.2 or 2.0 to 10, 25, 50,75, 100, 150, 200 or 300 mg of the compound disclosed herein, togetherwith a pharmaceutically acceptable diluent or carrier therefor.

Pharmaceutical compositions comprising Compounds of the Invention may beprepared using conventional diluents or excipients and techniques knownin the galenic art. Thus oral dosage forms may include tablets,capsules, solutions, suspensions and the like.

Methods of Making Compounds of the Invention

The compounds of the Invention and their pharmaceutically acceptablesalts may be made using the methods as described and exemplified hereinand/or by methods similar thereto and/or by methods known in thechemical art. Such methods include, but not limited to, those describedbelow. If not commercially available, starting materials for theseprocesses may be made by procedures, which are selected from thechemical art using techniques which are similar or analogous to thesynthesis of known compounds.

Various NEP inhibitors and starting materials therefor may be preparedusing methods described in US 2006-0041014 A1, EP 1097719 A1, U.S. Pat.No. 8,513,244, and US 2013-0330365 A1. All references cited herein arehereby incorporated by reference in their entirety.

Various starting materials and/or Compounds of the Invention may beprepared using methods described in WO 2006/133261 and WO 2009/075784.All references cited herein are hereby incorporated by reference intheir entirety.

Terms and Abbreviations:

-   -   DMF=N,N-dimethylforamide,    -   MeOH=methanol,    -   THF=tetrahydrofuran,    -   equiv.=equivalent(s),    -   h=hour(s),    -   HPLC=high performance liquid chromatography,    -   LiHMDS=lithium bis(trimethylsilyl)amide,    -   NMP=1-methyl-2-pyrrolidinone,    -   Pd₂(dba)₃=tris[dibenzylideneacetone]dipalladium(0)    -   TFA=trifluoroacetic acid,    -   TFMSA=trifluoromethanesulfonic acid

EXAMPLES Example 14-((7-Isobutyl-5-methyl-4,6-dioxo-3-(phenylamino)-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide

A mixture of7-isobutyl-5-methyl-3-(phenylamino)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(50 mg, 0.18 mmol), 4-(bromomethyl)benzenesulfonamide (48 mg, 0.19 mmol)and potassium carbonate (26.5 mg, 0.19 mmol) in DMF (1 mL) is stirred atroom temperature for 4 days. After filtration, the filtrate is purifiedwith a semi-preparative HPLC system equipped with a reversed-phase C18column using a gradient of 0-75% acetonitrile in water containing 0.1%formic acid over 16 min to give 41.4 mg of product as a white solid(HPLC purity: 98%; Yield: 54%). 1H NMR (500 MHz, Chloroform-d) δ 7.79(d, J=8.4 Hz, 2H), 7.29 (t, J=7.9 Hz, 2H), 7.13 (t, J=7.4 Hz, 1H), 7.08(d, J=8.4 Hz, 2H), 6.91 (d, J=7.5 Hz, 2H), 6.88 (s, 1H), 4.98 (s, 2H),4.76 (s, 2H), 3.82 (d, J=7.5 Hz, 2H), 3.34 (s, 3H), 2.41-2.20 (m, 1H),0.97 (d, J=6.7 Hz, 6H). MS (ESI) m/z 483.1 [M+H]⁺.

Example 24-((7-isobutyl-5-methyl-4,6-dioxo-3-(phenylamino)-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzoicacid

A mixture of7-isobutyl-5-methyl-3-(phenylamino)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(50 mg, 0.18 mmol), methyl 4-(bromomethyl)benzoate (44 mg, 0.19 mmol)and potassium carbonate (44.2 mg, 0.32 mmol) in DMF (1 mL) is stirred atroom temperature overnight. The mixture is diluted with DMF (1 mL)followed by adding 2.5 N NaOH aqueous solution (2 mL), and then heatedat 75° C. for 2 h. The mixture is neutralized with concentrated HCl andthen evaporated to dryness under reduced pressure. The residue istreated with DMF and then filtered with a 0.45 μm microfilter. Thefiltrate is purified with a semi-preparative HPLC system equipped with areversed-phase C18 column using a gradient of 0-85% methanol in watercontaining 0.1% formic acid over 16 min to give 28 mg of product as awhite solid (HPLC purity: 97%; Yield: 39%). 1H NMR (500 MHz,Chloroform-d) δ 7.95 (d, J=8.4 Hz, 2H), 7.31-7.24 (m, 2H), 7.11 (t,J=7.4 Hz, 1H), 7.02 (d, J=8.4 Hz, 2H), 6.93-6.85 (m, 3H), 4.99 (s, 2H),3.83 (d, J=7.5 Hz, 2H), 3.35 (s, 3H), 2.41-2.22 (m, 1H), 0.97 (d, J=6.7Hz, 6H). MS (ESI) m/z 448.2 [M+H]⁺.

Example 34-((3-(4-Fluorophenylamino)-5-methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide

(a)4-((5-Methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide

A mixture of5-methyl-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (708mg, 3.0 mmol), 4-(bromomethyl)benzenesulfonamide (750 mg, 3.0 mmol) andpotassium carbonate (1.24 g, 9.0 mmol) in DMF (15 mL) is stirred at roomtemperature for 6 h. The mixture is diluted with water (120 mL) and thenextracted with 10% MeOH in CH₂Cl₂ five times. The combined organic phaseis evaporated to dryness to give 1.4 g of crude product as an off-whitesolid, which is used in the next step without further purification. MS(ESI) m/z 406.2 [M+H]⁺.

(b)4-((3-Chloro-5-methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide

To a solution of the crude4-((5-methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide(1.36 g) and hexachloroethane (2.38 g, 10.1 mmol) in CH₂Cl₂ (5 mL) isadded 1.0 M LiHMDS in THF (16 mL, 16 mmol) dropwise. After thecompletion of the addition, the mixture is stirred at room temperaturefor 30 min and then poured into cold water (120 mL), followed byextractions with CH₂Cl₂ (25 mL×5). The combined organic phase is washedwith brine (25 mL×2) and then evaporated to dryness. The residue ispurified on a silica gel column using a gradient of 0% to 65% ethylacetate in hexanes to give 350 mg of product as an off-white solid. MS(ESI) m/z 440.1 [M+H]⁺.

(c)4-((3-(4-Fluorophenylamino)-5-methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide

A suspension of4-((3-chloro-5-methyl-7-neopentyl-4,6-dioxo-4,5,6,7-tetrahydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)benzenesulfonamide(100 mg, 0.227 mmol), 4-fluorobenzenamine (50 mg, 0.45 mmol) andpotassium carbonate (94 mg, 0.68 mmol) in tert-amyl alcohol (1.0 mL) isdegassed with argon and then Xantphos (6.6 mg, 0.011 mmol) and Pd₂(dba)₃(5.3 mg, 0.0058 mmol) are added. The suspension is degassed again andthen heated at 110° C. for 36 h. After cooled to room temperature, themixture is diluted with saturated NaHCO₃ aqueous solution (100 mL) andthen extracted with CH₂Cl₂ (25 mL×3). The combined organic phase isevaporated to dryness under reduced pressure. The obtained residue istreated with DMF and then filtered with a microfilter. The filtrate ispurified with a semi-preparative HPLC system using a gradient of 0-53%acetonitrile in water containing 0.1% formic acid over 16 min to give 17mg of the final product as an off white solid (HPLC purity: 97%). 1H NMR(500 MHz, Chloroform-d) δ 7.78 (d, J=8.3 Hz, 2H), 7.64 (s, 1H),7.57-7.49 (m, 2H), 7.13 (d, J=8.1 Hz, 2H), 7.01 (t, J=8.7 Hz, 2H), 5.75(br, 2H), 5.55 (s, 2H), 3.84 (s, 2H), 3.37 (s, 3H), 0.99 (s, 9H). MS(ESI) m/z 515.2 [M+H]+.

Example 4 Measurement of PDEI Inhibition In Vitro Using IMAPPhosphodiesterase Assay Kit

Phosphodiesterase 1 (including PDE1A, PDE1B and PDE1C) is acalcium/calmodulin dependent phosphodiesterase enzyme that convertscyclic guanosine monophosphate (cGMP) to 5′-guanosine monophosphate(5′-GMP). PDEIB can also convert a modified cGMP substrate, such as thefluorescent molecule cGMP-fluorescein, to the correspondingGMP-fluorescein. The generation of GMP-fluorescein from cGMP-fluoresceincan be quantitated, using, for example, the IMAP (Molecular Devices,Sunnyvale, Calif.) immobilized-metal affinity particle reagent.

Briefly, the IMAP reagent binds with high affinity to the free5′-phosphate that is found in GMP-fluorescein and not incGMP-fluorescein. The resulting GMP-fluorescein-IMAP complex is largerelative to cGMP-fluorescein. Small fluorophores that are bound up in alarge, slowly tumbling, complex can be distinguished from unboundfluorophores, because the photons emitted as they fluoresce retain thesame polarity as the photons used to excite the fluorescence.

In the phosphodiesterase assay, cGMP-fluorescein, which cannot be boundto IMAP, and therefore retains little fluorescence polarization, isconverted to GMP-fluorescein, which, when bound to IMAP, yields a largeincrease in fluorescence polarization (Amp). Inhibition ofphosphodiesterase, therefore, is detected as a decrease in Δmp.

Enzyme Assay

Materials: All chemicals are available from Sigma-Aldrich (St. Louis,Mo.) except for IMAP reagents (reaction buffer, binding buffer, FL-GMPand IMAP beads), which are available from Molecular Devices (Sunnyvale,Calif.).

Assay: Phosphodiesterase enzymes that may be used include:3′,5′-cyclic-nucleotide-specific bovine brain phosphodiesterase (Sigma,St. Louis, Mo.) (predominantly PDEIB but also contains PDE1A and PDE1C)and recombinant full length human PDE1A, PDE1B and PDE1C which may beproduced e.g., in HEK or SF9 cells by one skilled in the art. The PDE1enzyme is reconstituted with 50% glycerol to 2.5 U/ml. One unit ofenzyme will hydrolyze 1.0 μmol of 3′,5′-cAMP to 5′-AMP per min at pH 7.5at 30° C. One part enzyme is added to 1999 parts reaction buffer (30 μMCaCl₂, 10 U/ml of calmodulin (Sigma P2277), 10 mM Tris-HCl pH 7.2, 10 mMMgCl₂, 0.1% BSA, 0.05% NaN₃) to yield a final concentration of 1.25mU/ml. 99 μl of diluted enzyme solution is added into each well in aflat bottom 96-well polystyrene plate to which 1 μl of test compounddissolved in 100% DMSO is added. The compounds are mixed andpre-incubated with the enzyme for 10 min at room temperature. The FL-GMPconversion reaction is initiated by combining 4 parts enzyme andinhibitor mix with 1 part substrate solution (0.225 μM) in a 384-wellmicrotiter plate. The reaction is incubated in dark at room temperaturefor 15 min. The reaction is halted by addition of 60 μl of bindingreagent (1:400 dilution of IMAP beads in binding buffer supplementedwith 1:1800 dilution of antifoam) to each well of the 384-well plate.The plate is incubated at room temperature for 1 hour to allow IMAPbinding to proceed to completion, and then placed in an Envisionmultimode microplate reader (PerkinElmer, Shelton, Conn.) to measure thefluorescence polarization (Amp).

A decrease in GMP concentration, measured as decreased Δmp, isindicative of inhibition of PDE activity. IC₅₀ values are determined bymeasuring enzyme activity in the presence of 8 to 16 concentrations ofcompound ranging from 0.0037 nM to 80,000 nM and then plotting drugconcentration versus Δmp, which allows IC₅₀ values to be estimated usingnonlinear regression software (XLFit; IDBS, Cambridge, Mass.).

The Compounds of the Invention are tested in an assay as described orsimilarly described herein for PDE1 inhibitory activity, which compoundsgenerally have IC₅₀ values against PDE1A (HEK) and/or PDE1C (HEK) ofless than 25 nM.

Example 5 Pharmacokinetic Analysis of the Compounds of the Invention

Animals: Male, C57BL/6 mice (Jackson Labs, 25-30 g in body weight) areprovided by Jackson Laboratories. Up to five mice are housed per cageand are maintained under a 12 hour light/dark cycle with access to foodand water ad libitum. All procedures for the handling and use of animalsfollow the guidelines of the Institutional Animal Care and Use Committee(IACUC) of Columbia University, in accordance with NIH guidelines. Eightweek-old mice (N=3/dose level or treatment group) are used in theexperiments.

Experimental Treatment: Compounds: Selected Compounds of the Inventionare evaluated in the present study. Formulation/Vehicle: 3% 1N HCl, 5%Labrasol and 92% of 5% TPGS in 0.05M Citrate buffer (CB, pH 4.0). Thetest compound(s) are prepared as solution in vehicle and are dosed in avolume of 8 ml/kg.

Compound Preparation: Powdered stocks of the test compound(s) aremeasured and dissolved in 3% 1N HCl, 5% Labrasol and 92% of 5% TPGS in0.05M Citrate buffer (CB, pH 4.0). Two or three layers of 3 mm glassbeads are added to the bottom of the 10 ml glass tube to promote mixing.The tube is mixed using a benchtop vortex mixer then sonicated using aVWR sonicator (model 750) for about 5 min until the drug powder istotally dissolved in into a vehicle solution.

Treatment of Animals: Mice (N=3 mice/dose/time point) receive a 10 mg/kgoral (PO) dose of the test compound(s) at time 0. Groups of mice arekilled at a specified time point, either 0.25, 0.5, 1, or 2 h after drugadministration. Brain tissue is collected and frozen at −80° C., untilanalysis. Blood is collected from the mice by puncture of theretro-orbital vein using a Pasteur pipette (VWR, Cat#53283-911), thendeposited into silicon-coated blood collection tubes containing 0.105Msodium citrate solution (BD Vacutainer, #366392, Franklin Lakes, N.J.).Blood samples are centrifuged at the speed of 8000 g for 40 minutes in4° C. (TOMY, refrigerated benchtop microcentrifuge, Fremont, Calif.94583) and plasma decanted into Eppendorf tubes and frozen at −80° C.until analysis. Plasma and brain tissue samples are processed andanalyzed by the analytical group using LC-MS/MS methods, as describedbelow.

Sample Preparation: Samples of plasma are prepared for analysis asfollows: 50 μL of the plasma samples is transferred into a 500 μlpolypropylene microtube (Eppendorf Cat#022363611) as follows:

Standards Samples 50 μL control (blank) plasma 50 μL test sample plasma10 μL standard working solution 10 μL 1:1 Methanol:Water in 1:1Methanol:Water 150 μL 0.1 μM Standard in 150 μL 0.1 μM Standard inMethanol Methanol

Each tube is vortex mixed, then centrifuged for 20 min at 15000 rpm. Thesupernatant is collected and 100 μL of each is then transferred into a96-well polypropylene Elisa plate for mass spectrometric analysis.

Samples of brain homogenate were prepared for analysis as follows:Approximately 0.5 g of brain tissue is weighed and homogenized with 1 mLMilli-Q water. Then 60 μL of the resulting homogenate is thentransferred into a clean 500 μL polypropylene microtube (EppendorfCat#022363611) and treated as shown below:

Standards Samples 60 μL control (blank) brain 60 μL test sample brainhomogenate homogenate 20 μL standard working solution 20 μL 1:1Methanol:Water in 1:1 Methanol:Water 180 μL 0.1 μM Standard in 180 μL0.1 μM Standard in Methanol Methanol

Each tube is vortexed, then centrifuged for 20 min at 15000 rpm using aTomy benchtop centrifuge at 4° C., Standard is an internal standard usedfor LC-MS/MS quantitation. 150 μL of each supernatant is thentransferred into a 96-well plate for mass spectrometric analysis. Anyremaining plasma or homogenate is stored at approximately −20° C.pending any necessary repeat analysis. For each test sample, acalibration curve is prepared covering the range of 0.5-500 ng/mL.

HPLC and Mass Spectrometric Analysis: Analysis to quantify theconcentration of each compound in plasma and brain homogenate is carriedout using reverse phase HPLC followed by mass spectrometric detectionusing the parameters listed:

HPLC: Waters Alliance 2795 HT

Mobile phase A: 0.1% Formic acid in water

Mobile phase B: 0.1% Formic acid in methanol

Column: Phenomenex Synergi 4μ A Fusion-RP 50×2 mm

Column Temperature: 40° C.

Time (min) Solvent A (%) Solvent B (%) Flow Rate (mL/min) 0 80 20 0.6 20 100 0.6 4 0 100 0.6

Waters Alliance 2795 LC rapid equilibration flow (mL/min): 5

Waters Alliance 2795 LC rapid equilibration time (min): 0.25

Re-equilibration time (min): 1

Injection volume (μL): 10

Each compound is detected and quantified using Multiple ReactionMonitoring (MRM) of positive electrospray mode with a WatersQuattroMicro™ mass spectrometry system.

RESULTS: Plasma and Brain Analysis: All the test compound(s) testedcould be detected and analyzed when spiked into control plasma. Standardcurves are established prior to the analysis of the samples and provedlinear over the range of 0.5-1500 ng/mL in plasma and 0.5-500 ng/mL inbrain. Plasma and brain levels of each compound are determined andexpressed as means±standard deviation for each compound at each timepoint. Brain and plasma C_(max) and T_(max) values are estimated foreach compound by visual inspection of the data. A ratio of brain/plasmaconcentration (B/P) is also calculated for each compound by dividingBrain AUC_((0-2h)/)Plasma AUC_((0-2h)).

Results: Using the procedure described or similarly described, thecompounds of Examples 1-2 are tested and display oral bioavailabilitywith low brain exposure (B/P ratios approximately 0.05 or below).

What is claimed is:
 1. A compound of Formula I:

in free or salt form, wherein (i) R₁ is —NH(R₄), wherein R₄ is phenyloptionally substituted with halo; (ii) R₂ is H or C₁₋₆ alkyl; (iii) R₃is —SO₂NH₂ or —COOH.
 2. The compound according to claim 1, wherein R₁ is—NH(R₄), and wherein R₄ is phenyl.
 3. The compound according to claim 1,wherein R₁ is —NH(R₄), and wherein R₄ is 4-fluorophenyl.
 4. The compoundaccording to claim 1, wherein R₂ is C₁₋₆alkyl.
 5. The compound accordingto claim 1, wherein R₂ is isobutyl.
 6. The compound according to claim1, wherein R₂ is neopentyl.
 7. The compound according to claim 1,wherein R₃ is SO₂NH₂.
 8. The compound according to claim 1, wherein R₃is —COOH.
 9. The compound according to claim 1 which is the compound:

in free or salt form.
 10. The compound according to claim 1 which is thecompound:

in free or salt form.
 11. The compound according to claim 1 which is thecompound:

in free or salt form.
 12. A combination comprising a compound accordingto claim 1, in free or pharmaceutically acceptable salt form, and one ormore other therapeutic agents useful for the treatment of cardiovasculardisorders, in free or pharmaceutically acceptable salt form.
 13. Apharmaceutical composition comprising a compound according to claim 1,in free or pharmaceutically acceptable salt form, in admixture with apharmaceutically acceptable diluent or carrier.
 14. A method for thetreatment of a disease or disorder which may be ameliorated bymodulating cGMP/PKG-dependent signaling pathways comprisingadministering to a patient in need thereof an effective amount of thecompound according to claim 1, in free or pharmaceutically acceptablesalt form; wherein the disease or disorder is congestive heart failure.15. The method according to claim 14, further comprising administering aPDE5 inhibitor, in free or pharmaceutically acceptable salt form.
 16. Apharmaceutical composition comprising the combination according to claim12, in admixture with a pharmaceutically acceptable diluent or carrier.